Zoogloea transformation using exopoly saccharide non-capsule producing strains

ABSTRACT

Two new bacterial strains designated Zoogloea ramigera 115SL and Zoogloea ramigera 115SLR, a rifampicin resistant derivative of 115SL, have been developed. These strains are derived from the wild type Zoogloea ramigera 115, ATCC 25935. The two new strains produce a novel exopolysaccharide (EPS) and have several desirable characteristics that are absent from the parent strain, including improved culture properties, since they do not produce an EPS capsule layer like that of the parent 115 strain. The 115SL EPS is instead excreted as a slime layer which is not confined to the immediate area surrounding the cells. Since cells are not trapped within a floc where they grow at a reduced rate or die because of nutrient starvation, the new strains have more consistent and reproducible growth cycles and increased growth rates. As a consequence, exopolysaccharide production is more consistent and titers are higher. The separation of the EPS from the cells is also much easier and more economical. The other very important characteristic of strains 115SL and 115SLR is that they are able to receive foreign DNA using conventional techniques due to the absence of the capsule layer. This facilitates the application of recombinant DNA technology to control and produce novel expolysaccharides.

BACKGROUND OF THE INVENTION

This application is a continuation-in-part application of U.S. Ser. No.891,136 filed July 28, 1986, by Anthony J. Sinskey, Donald DavidsonEasson Jr., Oliver P. Peoples and Chokyun Rha entitled "Method toControl and Produce Novel Biopolymers". now abandoned in favor ofpending continuation application U.S. Ser. No. 07/329,594 filed Mar. 28,1989.

The present invention is in the field of genetically engineeredorganisms, particularly genetically modified exopolysaccharide producingstrains of Zoogloea ramigera.

Polysaccharide biopolymers have found applications in many industries,including the food, cosmetic, chemical, biomedical, waste treatment andoil industries.

Flocculation is one important commercial use of biopolymers. Severaltypes of floc-forming bacteria have been identified. The most efficientare the cellulose, or cellulose-like, producing bacteria such as certainspecies of Pseudomonas, Aerobacter, Agrobacterium, Azotobacter andZoogloea. Flocculation of these bacteria appears to occur when cellsbecome embedded in a network of exopolysaccharide fibrils. Otherfloc-forming bacteria produce capsular polysaccharides enclosing largepackets of cells which lead to floc formation. An example of thisphenomenon occurs with Zoogloea ramigera 115. In some cases theseexocellular polysaccharides have metal ion binding properties.

Z. ramigera 115, designated ATCC 25935 by the American Type CultureCollection, Rockville, Md., is an exopolysaccharide matrix formingstrain which, when grown in a nitrogen limiting medium, converts 60%(w/w) of the available glucose substrate into a water soluble capsularbranched heteropolysaccharide composed of glucose and galactose in amolar ratio of 2:1 with approximately 3% to 5% pyruvate. The negativelycharged carboxyl groups of the pyruvate are thought to be primarilyresponsible for the biopolymer's high affinity for heavy metal ions.

Due to the unique rheological and strong metal binding properties of theZoogloea ramigera exocellular polysaccharide, it is desirable toisolate, characterize, express and modify the exopolysaccharide genesproduced by Zoogloea ramigera strains. It is also desirable to providethe genes or complementary nucleotide sequences for production ofexocellular polysaccharides produced by Zoogloea ramigera for use insynthesis of novel polysaccharides.

It is therefore an object of the present invention to provide nucleotidesequences which can be used to produce novel biopolymers, especially theZoogloea ramigera exopolysaccharides.

It is still further object of the present invention to produce a systemfor production of biopolymers with enhanced rate and level of synthesisand greater ease in isolation of the biopolymers.

SUMMARY OF THE INVENTION

Two new bacterial strains designated Zoogloea ramigera 115SL andZoogloea ramigera 115SLR have been developed. They have been designatedATCC 53589 and ATCC 53590, respectively, as deposited on Mar. 5, 1987with the American Type Culture Collection in Rockville Md. Strain 115SLRis a rifampicin resistant derivative of 115SL. These strains are derivedfrom the wild type Zoogloea ramigera 115, ATCC 25935. The two newstrains produce a novel exopolysaccharide (EPS) and have severaldesirable characteristics that are absent from the parent strain.

The new strains, 115SL and 115SLR, have improved culture properties, ascompared to the parent 115 strain, since they do not produce an EPScapsule layer like that of strain 115. The 115SL EPS is excreted as aslime layer which is not confined to the immediate area surrounding thecells. The result is that strains 115SL and 115SLR do not flocculateduring growth, unlike 115 which forms large cell flocs during the growthphase. Since cells are not trapped within a floc where they grow at areduced rate or die because of nutrient starvation, the new strains havethe advantage of more consistent and reproducible growth cycles andincreased growth rates.

As a consequence of the higher cell density and healthier cultures,exopolysaccharide production is more consistent and titers are higher.The separation of the EPS from the cells is also much easier and moreeconomical.

In contrast to strain 115, a very important characteristic of strains115SL and 115SLR is that they are able to receive foreign DNA usingconventional techniques such as by conjugation, due to the absence ofthe capsule layer. This facilitates the application of recombinant DNAtechnology to control and produce novel expolysaccharides.

Compositional analysis of the exopolysaccharide produced by 115SL and115SLR demonstrates that there are no differences between their EPS andthe EPS of strain 115 with respect to monosaccharide composition.However, there is an increase of about 30 to 50% in the pyruvate contentin the EPS from 115SL and 115SLR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the derivation of Z. ramigera strains115SL and 115SLR.

FIG. 2 is a photo of Z. ramigera 115 (right) and Z. ramigera 115 SL(left), demonstrating the floc-forming characteristic of strain 115 andits absence in strain 115SL which produces a turbid or non-flocculatedculture.

FIGS. 3A and B are graphs of the growth (measured as mg protein/l) andexopolysaccharide production, measured as mg exopolysaccharide/lreleased into the supernatant and the total mg exopolysaccharide/lproduced, of Z. ramigera 115 (A) and Z. ramigera 115SL (B).

DETAILED DESCRIPTION OF THE INVENTION

Isolation of new Z. ramigera strains 115SL and 115SLR and identificationof their genes for polysaccharide synthesis enable development ofstrategies for the manipulation and control of the biosynthetic pathwayfor exopolysaccharide production at the genetic level. Strategies forcontrolled polymer production and structure include: placing thepolysaccharide biosynthetic genes under the control of regulatablepromoters; the introduction of these genes into new host stains toenable the development of more economic processes for polysaccharideproduction; mutagenesis of the genes to alter the enzyme activities andthe resulting polymer structure; and the construction of novel pathwaysfor polysaccharide synthesis by "mixing" genes from different strains ofZ. ramigera or other organisms.

The novel Z. ramigera strains were derived from Z. ramigera 115 (ATCC25935) obtained from the American Type Culture Collection Rockville,Maryland, as shown in FIG. 1. The Z. ramigera 115 strain was mutated byaddition of 40 micrograms nitrosoguanidine (NTG)/ml of cells followed byincubation without shaking at 30° C. Samples were treated for betweenzero and 60 minutes to establish a kill curve (concentration of cellsdecreased from 1×10⁷ at zero minutes to 6.4×10⁵ at 60 minutes). Thecells that were treated for twenty minutes (surviving concentrationequal to 6×10⁶ cells) to thirty minutes (surviving concentration equalto 4×10⁶ cells) were then plated out. Cells were screened on platescontaining Celluflor for loss of fluorescence, indicating loss ofpolysaccharide production, and a change in morpholopy.

The Zoogloea ramigera strains were characterized by microscopy,morphological characterization and determination of the ability to growand produce polysaccharide on different mediums. A complex medium and adefined medium were selected that contain all the requirements forgrowth and polysaccharide production and detection.

The media and culture conditions are as follows: Z. ramigera culturesare stored frozen at -70° C. in trypticase soy broth (TSB) mediumcontaining 7% DMSO and 15% glycerol. The various Z. ramigera strains areroutinely cultured in either the TSB medium or a defined medium,described by Norberg and Enfors in Appl. Env. Microbiol. 44, 1231-1237(1982) having the following composition: 25 g glucose, 2 g K₂ HPO₄, 1 gKH₂ PO₄, 1 g NH₄ Cl; 0.2 g MgSO₄ 7H₂ O; 0.01 g yeast extract (DifcoLaboratories) in one liter distilled water, where the glucose, MgSO₄ 7H₂O, yeast extract and salts are autoclaved separately. 100 ml cultures ofZ. ramigera are grown on a rotary shaker (200 rpm) at 30° C. in 500 mlbaffled shake flasks for periods up to two weeks.

Cellufluor (Polysciences Chemicals, Warrington, Pa.) is a fluorescentdye, disodium salt of4,4'-bis-[4-anilino-bis-diethyl-amino-S-triazin-2-ylamin]-2,2'-stilbene-disulfonic acid, that binds specifically to beta (1-3) andbeta (1-4) glycosyl linkages and fluoresces when exposed to UV light.Cellufluor is added to agar plates, pH 7.4, at a concentration of 200micrograms/ml and used to determine polysaccharide production in Z.ramigera. Strain 115 flocculates and has a discernable polysaccharidecapsule layer surrounding the cell flocs, as shown in FIG. 2, as well asa very unique colonial morphology. As shown in FIG. 1, the change inappearance is striking. The 115SL and 115SLR strains of Z. ramigera donot flocculate.

115SLR is a spontaneous rifampicin resistant mutant of 115SL and isparticularly useful in selection processes during genetic manipulations.For example, it can be used to select against donor strains inexperiments involving the conjugal transfer of plasmids and plasmid/genelibraries.

Southern hybridization of ³² P-pHP27, a 115/pLAFR3 recombinant plasmidthat causes a change in the morphology of Z. ramigera I-16-M, was usedto confirm that 115SL is derived from 115.

The 115 polymer is purified by the addition of concentrated NaOH to thecell culture at a final concentration of 0.2M, followed by the additionof 3 volumes of ethanol to precipitate the polymer and other materials.The precipitate is collected and redissolved in half the original volumeof water. Protein is removed by either extracting twice with phenol orby ultrafiltration. The aqueous phase is dialyzed, lyophilized andground to yield a fine white powder.

Since it is not cell-bound, the exopolysaccharide produced by 115SL and115SLR is purified without using the alkali treatment or the phenolextraction. Not only is the purification process thereby simplified, itdoes not remove alkali-labile acetyl moieties from the purified 115SLand 115SLR exopolysaccharide. Since all previously used purificationprocedures have included an alkali step, this is believed to be thefirst time the exopolysaccharide has been available in purifed form withacetate groups present on the polymer.

Total carbohydrate concentration in culture broths and polymer solutionsis determined by the phenol reaction, described by Gerhardt in Manual ofMethods for General Bacteriol. (Washington Amer. Soc. Microbiol. 1981).Glucose, galactose and Xanthan gum (Sigma Chemical Co., St. Louis, Mo.)are used as standards. Total protein concentration in culture broths andpolymer solutions is determined using the Bio-Rad protein assay (Bio-RadLaboratories, Richmond, Calif., 1979). Lysozyme is used as the standard.Cellular protein is released by boiling in 0.2N NaOH.

Purified polysaccharide is hydrolyzed in 1M trifluoroacetic acid at 120°C. for times varying between 30 minutes and 2 hours. Monosaccharides inthe polysaccharide hydrolysate are separated using a Waters HPLCequipped with a Brownlee Polypore PB, lead loaded cation exchangecolumn, operated at 85° C., with water as the eluent. Detection is byrefractive index using a Waters Model 401 Differential Refractometer.The polysaccharide can further characterized by proton NMR spectroscopyand infared spectroscopy. Infrared analysis, along with themonosaccharide composition data, can be compared to the composition andIR scans of polysaccharides from mutant or genetically manipulatedstrains to detect changes in structure.

The polymer produced by Zoogloea ramigera 115SL consists of glucose andgalactose in a ratio of approximately 2:1, the same as theexopolysaccharide produced by strain 115. The pyruvate content isslightly higher in the 115SL and 115SLR strains, however, byapproximately 30 to 50%.

The isolated exopolysaccharide produced by Z. ramigera 115 SL and 115SLR has a number of uses similar to that of the exopolysaccharideisolated for the parent strain. For example, the polymer can be used asa flocculant, in the isolation and removal of heavy metal ions, and as aviscosity modifying agent. However, although similar to the 115exopolysaccharide, some differences in application are expected due tothe slightly higher pyruvate level and the presence of the acetatemoiety.

The new strains, 115SL and 115SLR, are useful in ways that the parentstrain 115 is not. For example, it produces higher, more consistentlevels of polysaccharide which is more easier purified since the cellfloc present in 115 is absent. The number of cells which can be grown inculture is also greater due to the easier diffusion of nutrients intothe cell. The differences in cell growth and polymer production aredemonstrated in FIGS. 3A and 3B. The total amount of EPS (mg/l)produced, as well as the total amount released into the medium over timeas a percentage of the total EPS produced (mg/l), for 115 (FIG. 3A) issignificantly less than that for 115SL (FIG. 3B). The initial growthcurve for 115SL (FIG. 3B), shown by the total protein, is also muchsharper than it is for 115 (FIG. 3B).

The primary advantage of the new 115SL and 115SLR strains, however, isthat they can be more easily manipulated genetically. As demonstratedbelow, the 115SL and 115SLR strains can accept foreign or plasmid DNA ina relatively high yield by conjugal transfer without extensivemanipulation. This allows not only the isolation, characterization andmodification of the genes for exopolysaccharide production but also theinsertion of genes encoding additional enzymes for synthesis ofbiopolymers. Methods for manipulation and construction of pathways forpolymer synthesis are described in detail in co-pending application.U.S. Ser. No. 07/3429,594 filed Mar. 28, 1989, now abandoned.

As an example of the application of these methods to Z. ramigera 115SLand 115SLR, transposon mutants of Z. ramigera 115SL were constructed bythe method described in pending, U.S. Ser. No. 07/329,594 filed Mar. 28,1989, now abandoned and screened for changes in exopolysaccharidecomposition and production, which describes the transfer of a Tn5containing derivative of pRK 2013 in Z. ramigera strains using E. coliMM294A by conventional techniques for conjugation. Several Tn5 mutantsthat were non-fluorescent on Cellufluor were recovered. Although notadsorbing Cellufluor, the mutants are producing exopolysaccharide. Thesepolymers have apparently undergone a structural or compositional changefrom that of the original exopolysaccharide.

A pLAFR3/115 gene library was then introduced into these mutants. Theresulting transconjugants were screened for fluorescence on Cellufluorand for changes in colony morphology. One class of complemented strainsregained the ability to fluoresce on Cellufluor. A second class ofcomplemented strains had colony morphology identical to that of thewild-type 115.

Modifications and variations of the present invention, a method andmeans for producing genetically manipulated biopolymers including theexopolysaccharides produced by Zoogloea ramigera strains, will beobvious to those skilled in the art from the foregoing detaileddescription. Such modifications and variations are intended to comewithin the scope of the following claims.

We claim:
 1. A method for introducing foreign DNA into Zoogloeacomprising:providing isolated nucleotide sequences, inserting saidisolated sequences into a vector capable of replicating in Zoogloea; andintroducing said vector containing said isolated sequences into anexopolysaccharide producing, non-capsule forming Zoogloea ramigerastrain.
 2. The method of claim 1 wherein said sequences are insertedinto said Zoogloea ramigera by conjugation.
 3. The method of claim 1wherein said Zoogloea ramigera strain is ATCC 53589, or a derivativethereof.
 4. The method of claim 1 wherein said Zooglea ramigera strainis ATCC 53590, or a derivative thereof.
 5. The method of claim 1 furthercomprising mutating capsule-forming Zoogloea ramigera to form theexopolysaccharide, non-capsule producing Zoogloea ramigera.
 6. Zoogloearamigera 115SL ATCC
 53589. 7. Zoogloea ramigera 115SLR ATCC
 53590. 8.The Zoogloea ramigera of claim 6 further comprising a vector capable ofreplication in Zoogloea.
 9. The Zoogloea ramigera of claim 7 furthercomprising a vector capable of replication in Zoogloea.
 10. A method formaking a Zoogloea ramigera strain comprising mutating capsule-formingZoogloea ramigera.
 11. The method for making an exopolysaccharide,non-capsule producing Zoogloea ramigera strain of claim 10 wherein theZoogloea is mutated using a chemical mutagen.
 12. An isolatedexopolysaccharide, non-capsule producing Zoogloea ramigera produced bymutation of a capsule forming Zoogloea ramigera strain.